This project is broadly concerned with how a cell can integrate different patterns of gene expression and coordinate diverse biochemical and physiological activities. Previous work has revealed the existence of two regulatory systems, each capable of broad pleiotropic effects. The regulatory system under study here is mediated by an unusual nucleotide (ppGpp) and is estimated to maximally control the expression of approximately half of the Escherichia coli enzymes. The second pleiotropic regulatory system is mediated by cyclic AMP and is thought to mediate regulation of about 5 percent of the cellular proteins. Present work concerns genetic definition of the bacterial chromosomal region encoding the enzyme responsible for ppGpp synthesis on the ribosomes in order to ask whether the synthesis of ppGpp is a cellular function essential to life and to construct mutants with excessively high and low levels of ppGpp for further studies. This year progress has been made to purify ribosomal genes in order to assess mechanisms of ppGpp regulation of genetic expression in a biochemically pure system at the transcription level.